Histone Deacetylase Inhibitory Activity and Antiproliferative Potential of New [6]-Shogaol Derivatives.

Twenty newly synthesized derivatives of [6]-shogaol (4) were tested for inhibitory activity against histone deacetylases. All derivatives showed moderate to good histone deacetylase inhibition at 100 µM with a slightly lower potency than the lead compound. Most potent inhibitors among the derivatives were the pyrazole products, 5j and 5k, and the Michael adduct with pyridine 4c and benzothiazole 4d, with IC50 values of 51, 65, 61 and 60 µM, respectively. They were further evaluated for isoform selectivity via a molecular docking study. Compound 4d showed the best selectivity towards HDAC3, whereas compound 5k showed the best selectivity towards HDAC2. The potential derivatives were tested on five cancer cell lines, including human cervical cancer (HeLa), human colon cancer (HCT116), human breast adenocarcinoma cancer (MCF-7), and cholangiocarcinoma (KKU100 and KKU-M213B) cells with MTT-based assay. The most active histone deacetylase inhibitor 5j exhibited the best antiproliferative activity against HeLa, HCT116, and MCF-7, with IC50 values of 8.09, 9.65 and 11.57 µM, respectively, and a selective binding to HDAC1 based on molecular docking experiments. The results suggest that these compounds can be putative candidates for the development of anticancer drugs via inhibiting HDACs.

1. Introduction

The acetylation of histones by histone acetyltransferases (HATs) and the deacetylation of histones by histone deacetylases (HDACs) support the epigenetic regulation of gene expression to a substantial degree [1,2]. Epigenetic changes play an important role in tumorigenesis. Epigenetic post-translational modifications such as lysine deacetylation are involved in the progression of cancer [3]. HDACs have key effects on various cellular functions such as the regulation of gene transcription and cell proliferation, differentiation and death [4,5]. Moreover, HDACs are overexpressed in many human cancer cells. Therefore, HDAC inhibitors have been extensively explored as epigenetic therapeutics for cancer [6,7,8,9,10]. Currently, four HDAC inhibitors, including vorinostat (1, Zolinza®), romidepsin (Istodax®), belinostat (Beleodaq®) and panobinostat (Farydak®), have been approved by the U.S. Food and Drug Administration (USFDA) and launched on the market for the treatment of multiple myloma [11,12,13,14]. In addition, another HDAC inhibitor, chidamide (Epidaza®), has been approved by the Chinese Food and Drug Administration (CFDA) for the treatment of hematologic cancer [15]. There are eighteen known HDACs, grouped into four classes based on sequence similarity [16]. Class I (HDAC1, 2, 3 and 8), class IIa (HDAC4, 5, 7 and 9), class IIb (HDAC6 and 10) and class IV (HDAC11) HDACs are zinc-dependent enzymes, whereas class III HDACs (SIRT1-SIRT7) require NAD+ for activity [17]. All four USFDA-approved HDAC inhibitors are hydroxamic acids and pan-inhibitors that non-selectively inhibit most of HDAC isoforms (HDAC1-11) [18,19]. This non-selective property might explain the side effects observed in clinic and then limit their use in cancer therapy [20]. Moreover, hydroxamic acid, a strong zinc chelator, shows metabolic and pharmacokinetic issues, including glucuronidation, sulfonation and enzymatic hydrolysis, that result in a short in vivo half-life [21]. Therefore, the search for developing potent HDAC inhibitors with isoform selectivity and minimum side effects continues [22,23,24,25]. Several non-hydroxamic HDAC inhibitors have been reported, such as hydroxycapsaicin (2), [6]-gingerol (3) and [6]-shogaol (4) (Figure 1) [26,27,28,29,30]. Even though these compounds possess less HDAC inhibitory potency than hydroxamic HDAC inhibitors, they caught our attention as natural-derived compounds with low toxicities [31]. Natural-derived [6]-shogaol (4) had received a lot of attention because of its variety of biological activities, such as anti-inflammatory, antioxidant, anticancer, antibacterial activities, and HDAC inhibitor [32]. In this study, we continued with the modifications of [6]-shogaol (4) to improve inhibitor enzyme binding and aimed to determine whether these compounds would be potent HDAC inhibitors and manifest antiproliferative activities against cancer cells, as well as maintaining their low toxicity. The [6]-shogaol derivatives were designed to increase inhibitor enzyme binding via van der Waals, hydrogen bonds, hydrophobic and hydrophilic bonds, and π–π interactions by introducing hydrazone, pyrazole, amino and imine moiety into the molecular skeleton of [6]-shogaol (4).

Figure 1

Structures and IC50 values of known histone deacetylase inhibitors.

2. Results and Discussion

The natural compound [6]-shogaol (4) was isolated from ginger as previously described [27]. The immino derivatives 4a–4e were prepared by reacting [6]-shogaol (4) with hydrazine hydrate, 4-hydrazinobenzoic acid, 2-hydrazinopyridine, 2-hydrazinobenzothiazole and 2-hydrazino-2-imidazoline in ethanol at room temperature (Scheme 1). Reactions of [6]-shogaol (4) with phenylhydrazines including phenylhydrazine, o-tolylphenylhydrazine, 2-methoxyphenylhydrazine, 4-methoxyphenylhydrazine, 2-fluorophenylhydrazine, 4-fluorophenylhydrazine, 3-chlorophenylhydrazine, 4-chlorophenylhydrazine, 3-nitrophenylhydrazine, 4-nitrophenylhydrazine, and 4-hydrazinobenzoic acid in ethanol at 80 °C provided the pyrazole derivatives 5a–5k. All derivatives were obtained in good yields. The formations of the pyrazole rings were confirmed by the 13C-NMR chemical shift of about 62 and 153 ppm for the C-6 and C-8 positions, respectively. Reactions of [6]-shogaol (4) with primary amines, including 2-aminothiophenol, 2-aminophenol and aniline, in ethanol at 80 °C provided the Michael-addition products 6a–6c. Interestingly, Michael immino adduct 7 was gained as the major product from reacting [6]-shogaol (4) with o-phenylenediamine in ethanol at 80 °C. The reaction showed that the amino group at the ortho-position reacts at the carbonyl group to provide a seven-membered ring. The formation of the seven-membered ring of 7 was confirmed by the chemical shift of 13C-NMR at 174.1 and 65.7 ppm.

Scheme 1

Synthesis of new [6]-shogaol derivatives.

The synthesized derivatives were tested for HDAC inhibition with a commercial HDAC assay kit. The results are summarized in Table 1. All derivatives showed slightly weaker % HDAC inhibitions than the lead compound. The immino derivatives 4c and 4d showed the best HDAC inhibitory activity among the immino derivatives, with IC50 values of 61 ± 0.92 and 60 ± 0.84 µM, respectively. The immino derivatives with aromatic groups 4a–4e showed stronger HDAC inhibitory activities than the Michael-addition products 6a–6c, 7. However, this Michael-adduct type of [6]-shogaol could become thymidylate kinase inhibitors [33].

Table 1

HDAC inhibitory activity of compounds at 100 µM.

Compounds	%HDAC Inhibition	Compounds	%HDAC Inhibition
TSA	92	5a	74
4	86	5b	56
4a	73	5c	52
4b	73	5d	67
4c *	80	5e	54
4d *	80	5f	77
4e	73	5g	60
6a	54	5h	68
6b	64	5i	77
6c	53	5j *	85
7	63	5k *	83

* IC50; 4c = 61 ± 0.92 µM, 4d = 60 ± 0.84 µM, 5j = 51 ± 0.82 µM, 5k = 65 ± 1.12 µM.

To study the structure–activity relationship (SAR), phenyl pyrazole derivatives with various substitution groups at the aromatic region, such as methyl, methoxy, fluoro, chloro, nitro, and carboxyl groups, were evaluated as HDAC inhibitors. The substitution phenylpyrazoles 5b–5h showed slightly weaker % HDAC inhibition than the phenylpyrazole 5a. Interestingly, the para-substitution derivatives showed highly potent HDAC inhibitors than ortho- and meta-substitution derivatives. The p-nitrophenyl, pyrazole 5j, and p-carboxylphenyl pyrazole 5k derivatives were the most potent inhibitors among the pyrazole derivatives with IC50 values of 51 ± 0.82 and 65 ± 1.12 µM, respectively.

The most potent derivatives, 4c, 4d, 5j and 5k, were further investigated as the isoform-selective inhibitors of the isoforms class I HDACs (HDAC1, HDAC2, HDAC3 and HDAC8). The results from the molecular docking experiment are shown in Table 2. The immino derivative 4d showed a better binding affinity with HDAC1 and HDAC3 isoforms than TSA. The major interaction of 4d and HDAC1 (Figure 2) consisted of two hydrogen bonds between 4d and Phe205 (2.9 Å) and Leu271 (2.7 Å). The side chain of 4d was inserted into the catalytic channel of HDAC1, binding the cofactor Zn2+ ion. The binding mode of 4d and HDAC3 is shown in Figure 3. The 4d-HDAC3 complex showed that a stronger inhibitor–enzyme interaction consists in two hydrogen bonds between 4d and Asp92 (2.1 Å) and Gly143 (3.3 Å) of HDAC3. The coordination of the benzotriazole group to the Zn2+ ion (3.2 Å) can also be observed. Compound 5k showed a selectivity to HDAC2. The 5k-HDAC2 complex showed a complete insertion of its aromatic ring into the active site pocket, with multiple contacts with the tubular channel. The major interaction of 5k and HDAC2 (Figure 4) consisted of three hydrogen bonds between 5k and Gly154 (2.7 Å), Gly143 (2.7 Å), Leu276 (3.1 Å), the π–π interaction with Phe210, as well as the coordination of the aromatic ring to the Zn2+ ion (2.8 Å). The most potent compound in vitro 5j showed selectivity to HDAC1. The 5j-HDAC1 complex shows that the stronger inhibitor enzyme interaction consists of three hydrogen bonds between 5j and Phe150 (2.7 Å), Cys151 (2.7 Å), Gly300 (2.8 Å), the π–π interactions with Phe150, His140, and His178; as well as the coordination of the aromatic ring to the Zn2+ ion (2.0 Å) (Figure 5).

Table 2

In silico histone deacetylase inhibitory activity of the selected compounds.

Cp	HDAC1			HDAC2
	∆G *	Ki **	Binding Residues	∆G *	Ki **	Binding Residues
TSA	−8.12	1.12	Zn, His28, Asp99, Gly149, Phe205	−8.75	0.39	Zn, Gly32, His33, Asp104, Gly154
4c	−6.80	10.36	Glu98, Asp99, Glu271	−6.60	14.45	Gly154, Tyr209, Phe210, Leu276
4d	−8.31	0.81	Zn, Gly149, Tyr204, Phe205, Leu271	−7.65	2.48	Gly32, Thr309, Ile310
5j	−8.24	0.92	Zn, Phe150, Cys151, Gly300,	−7.05	6.79	Asn26, Tyr27, His38, Arg311
5k	−6.02	38.39	Tyr201, Leu211, Pro227, Lys361	−7.72	2.28	Zn, Gly143, Gly154, Leu276
Cp	HDAC3			HDAC8
	∆G *	Ki **	Binding Residues	∆G *	Ki **	Binding Residues
TSA	−8.23	0.93	Zn, His22, Gly143, Phe144, His172, Phe200	−8.85	0.33	Zn, His143, Phe152, Phe207, Phe208
4c	−7.60	2.67	Pro23, Asp93, Arg265, Leu266	−7.12	5.36	Phe152, Gly206, Phe207, Met274
4d	−9.18	0.19	Zn, His22, Asp92, Gly143, Phe200	−7.99	1.40	Phe152, Gly206, Phe207, Met274
5j	−7.42	3.64	Pro23, Phe199, Leu266	−7.45	3.47	Lys33, Pro273, Asn307, Leu308
5k	−6.69	12.42	Thr298, Glu300, Tyr331	−6.95	8.11	Lys33, Asn307, Leu308

* Free energy of binding (ΔG, kcal/mol), ** Inhibition constant (Ki, µM).

Figure 2

Interaction of 4d with HDAC1. Image was generated by DiscoveryStudio4.5.

Figure 3

The interaction modes 4d in the active site of HDAC3.

Figure 4

The interaction mode of 5k in the active site of HDAC2.

Figure 5

The interaction mode of 5j in the active site of HDAC1.

The in vitro HDAC inhibitions of the obtained compounds were carried out with the assay kit containing mixed-HDAC isoforms. In order to further investigate the isoform selectivity of the four most potent HDAC inhibitors, an in silico experiment was performed with each HDAC isoform. Therefore, the results were not fully related. However, compounds with good binding affinities should have the potential to be specific HDAC inhibitors for each isoform.

To complete the evaluation of these potent HDAC inhibitors, antiproliferative activities were determined in human cervical cancer (HeLa), human colon cancer (HCT116), human breast adenocarcinoma cancer (MCF-7), and cholangiocarcinoma (KKU-100 and KKU-M213B) cells. The results, as shown in Table 3 and Table 4, indicate that the derivatives 4c, 4d, 5j and 5k were less toxic to non-cancer cells than the lead compound. Compound 5j showed the best activity against HeLa, HCT116 and MCF-7, with IC50 values of 8.09, 9.65 and 11.57 µM, respectively. Additionally, compound 5j displayed a strong antiproliferative activity against cholangiocarcinoma cells, as shown in Table 4. Compounds 4c and 4d exhibited antiproliferative activity against cholangiocarcinoma cells with selectivity towards KKU-100 and KKU-M213B, respectively. Compound 5k, the least toxic compound, showed a good activity against HeLa cells with an IC50 value of 23.14 µM. Moreover, compound 5k appeared to be about 5-fold more selective towards HeLa cells than non-cancer cells, whereas [6]-shogaol (4) exhibited only a 3-fold selectivity.

Table 3

Antiproliferative activities of potent HDAC inhibitors against non-cancer and cancer cell lines.

Cpd	IC50 Values (Mean ± SD; n = 3 (μM))
	Vero Cells			HeLa Cells			HCT116 Cells			MCF-7 Cells
	24 h	48 h	72 h	24 h	48 h	72 h	24 h	48 h	72 h	24 h	48 h	72 h
Cisplatin	42.86 ± 2.39	12.73 ± 0.63	6.55 ± 0.81	17.07 ± 1.00	9.97 ± 0.34	6.45 ± 0.12	20.98 ± 2.56	7.61 ± 0.61	4.98 ± 0.77	29.17 ± 1.35	13.75 ± 0.54	10.42 ± 0.25
4	37.63 ± 0.94	35.96 ± 0.95	23.34 ± 0.24	16.86 ± 0.80	9.26 ± 0.46	8.10 ± 0.29	17.30 ± 0.57	12.37 ± 0.15	12.30 ± 0.13	24.78 ± 0.66	16.54 ± 0.15	13.39 ± 0.07
4c	69.47± 3.94	49.31 ± 0.18	45.17 ± 0.49	49.31 ±1.27	33.35 ±1.09	29.31 ±0.88	20.19 ±0.46	18.42 ±0.32	17.25 ±0.23	46.42 ±1.30	39.40 ±0.68	27.07 ±0.47
4d	169.6 ± 6.98	69.97 ± 1.27	60.15 ± 1.69	51.56 ± 1.92	37.23 ± 1.33	33.66 ± 0.76	40.08 ± 1.47	28.28 ± 0.79	26.63 ± 0.15	103.5 ± 3.17	57.81 ± 2.37	25.52 ± 1.15
5j	41.63 ± 2.99	32.20 ± 1.31	26.08 ± 0.47	26.83 ± 0.62	11.59 ± 0.60	8.09 ± 0.03	42.72 ± 2.23	10.18 ± 0.43	9.65 ± 0.17	27.85 ± 0.86	13.75 ± 0.56	11.57 ± 0.44
5k	143.0 ± 2.15	127.6 ± 1.73	116.7 ± 1.13	83.82 ± 1.83	56.78 ± 1.24	23.14 ± 0.76	94.44 ± 2.70	39.22 ± 1.06	34.47 ± 0.66	56.61 ± 3.21	50.54 ± 1.64	31.76 ± 0.88

Table 4

Antiproliferative activities of potent HDAC inhibitors against H-69 and cholangiocarcinoma cell lines.

Cpd	IC50 Values (Mean ± SD; n = 3 (µM))
	H-69 Cells			KKU-100 Cells			KKU-M213B Cells
	24 h	48 h	72 h	24 h	48 h	72 h	24 h	48 h	72 h
Cisplatin	>20	>20	15.80 ± 0.35	17.32 ± 0.12	11.18 ± 0.14	7.57 ± 0.07	>20	18.56 ± 0.57	12.98 ± 0.61
4	38.35 ± 0.84	31.98 ± 0.55	24.78 ± 0.24	27.64 ± 0.26	18.92 ± 0.48	17.04 ± 0.04	17.95 ± 0.05	12.92 ± 0.47	9.91 ± 0.17
4c	40.03 ± 1.57	30.63 ± 0.82	28.72 ± 0.36	36.82 ± 0.89	29.70 ± 0.96	19.20 ± 0.41	39.00 ± 0.27	28.98 ± 0.26	25.94 ± 0.71
4d	51.42 ± 1.51	41.44 ± 0.61	29.40 ± 0.89	41.63 ± 1.24	31.50 ± 0.80	25.75 ± 0.20	39.84 ± 0.29	20.60 ± 0.18	18.18 ± 0.75
5j	44.06 ± 0.89	34.63 ± 0.41	26.37 ± 0.43	27.22 ± 0.59	22.43 ± 0.65	17.45 ± 0.14	23.86 ± 0.85	19.30 ± 0.90	13.80 ± 0.16
5k	68.71 ± 1.15	52.67 ± 1.73	47.56 ± 0.73	68.14 ± 1.98	38.90 ± 0.65	26.99 ± 0.08	63.16 ± 2.52	35.68 ± 1.40	29.13 ± 1.17

Anticancer activities of the best-four HDAC inhibitors were investigated upon the proliferation of five human cancer cell lines in a time-dependent manner. All selected compounds showed an antiproliferative activity in the growth inhibition of cancer cell lines. The results support the use of these HDAC inhibitors as potential anticancer candidates.

3. Conclusions

In conclusion, a series of new immino and pyrazole derivatives of [6]-shogaol were designed and synthesized as potential HDACs inhibitors. All derivatives exhibited HDAC inhibitory activity in the micromolar concentration ranges. Among these derivatives, pyrazole 5j with p-nitro substituent displayed the most remarkable HDACs inhibitory activities. Additionally, pyrazole 5j showed the best activity against all tested cancer cells among the synthesized derivatives and the most selective binding to HDAC1 based on molecular docking experiments. Therefore, the in vivo experiments of 5j will be further investigated.

4. Experimental Section

4.1. General

Reagents were purchased from commercial sources (Sigma-Aldrich, Merck, and Carlo Erba). Reactions were monitored using analytical TLC plates (Merck, silica gel 60 F254), and compounds were visualized under ultraviolet light. Silica gel grade 60 (230–400 mesh, Merck) was used for column chromatography. The NMR spectra were recorded in the indicated solvents on a Varian Mercury Plus spectrometer operated at 400 MHz (1H) or 100 MHz (13C). The IR spectra were obtained on Perkin Elmer Spectrum One FT-IR spectrophotometer. Mass spectra were determined using a Micromass Q-TOF 2 hybrid quadrupole time-of-flight (Q-TOF) mass spectrometer with a Z-spray ES source.

4.2. Plant Material

The dried rhizome powder of ginger was obtained from the local market in Khon Kaen province, Thailand. The extraction and isolation of [6]-shogaol (4) followed previously described methods [27].

4.3. Structural Modifications

4.3.1. Synthesis of the Immino Derivatives (4a–4e)

To a solution of [6]-shogaol (4) (113 mg, 0.41 mmol) in ethanol (5 mL), NH2NH2.2HCl (64 mg, 0.76 mmol) was added. The solution was stirred at room temperature until completion based on TLC. The mixture was filtered, and the residue was washed with ethanol (10 mL) and dried with anhydrous sodium sulfate. Evaporation of the combined solvents gave a crude product. Purification of the crude product by column chromatography (100% dichloromethane) gave the yellow oil of compound 4a (90 mg, 0.31 mmol, 76%).

4-((3Z,4E)-3-Hydrazonodec-4-en-1-yl)-2-methoxyphenol (4a). 1H NMR spectrum of 4a is shown in Supplementary Materials. Yellow oil; yield: 76%; R = 0.50 (100% dichlorometane); IR (neat) υmax 3336 (OH), 1604 (C=N), 1515 (C=C), 1278 (C-O) cm−1.1H NMR (CDCl3, 400 MHz) δ 6.82 (dd, J = 2.0, 8.0 Hz, 1H), 6.69 (m, 2H), 6.09 (m, 1H), 5.48 (s, 1H), 3.87 (s, 3H), 2.83 (m, 2H), 2.75 (m, 2H), 2.69 (t, J = 8.0 Hz, 1H), 2.60 (q, J = 8.0 Hz, 1H), 1.42 (m, 2H), 1.25 (m, 4H), 0.87 (m, 3H). 13C NMR (CDCl3, 100 MHz) δ 149.1, 147.9, 146.4, 143.8, 133.2, 130.3, 120.8, 114.3, 111.0, 55.9, 42.0, 32.5, 29.2, 28.1, 23.8, 22.5, 14.1. HRMS-ESI (m/z) [M + Na]+ calcd for C17H26N2O2Na 313.1892, found 313.1882.

The same procedure was carried out with 4-hydrazinobenzoic acid hydrochloride, 2-hydrazinopyridine, 2-hydrazinobenzothiazole, and 2-hydrazino-2-imidazoline to convert [6]-shogaol (4) into 4b–4e.

4-(2-((3Z,4E)-1-(4-Hydroxy-3-methoxyphenyl)dec-4-en-3-ylidene)hydrazinyl)benzo-ic acid (4b). Yellow viscous liquid; yield: 75%; R = 0.45 (100% dichlorometane); IR (neat) υmax 3336 (OH), 1692 (C=O), 1603 (C=N), 1546 (C=C), 1268 (C-O) cm−1. 1H NMR (CDCl3, 400 MHz) δ 8.07 (d, J = 8.0 Hz, 2H), 7.25 (d, J = 8.0 Hz, 2H), 6.79 (d, J = 8.0 Hz, 1H), 6.62 (d, J = 8.0 Hz, 1H), 6.45 (d, J = 4.0 Hz, 1H), 6.40 (dd, J = 4.0, 8.0 Hz, 1H), 6.11 (s, 1H), 3.69 (s, 3H), 2.73 (t, J = 8.0 Hz, 2H), 2.59 (t, J = 8.0 Hz, 2H), 1.64 (m, 2H), 1.34 (m, 4H), 0.90 (m, 3H). 13C NMR (CDCl3, 100 MHz) δ 161.5, 154.2, 147.4, 144.5, 144.4, 141.9, 131.9, 130.2, 124.9, 120.5, 114.7, 114.6, 111.6, 105.1, 55.1, 34.8, 31.3, 29.1, 28.0, 27.6, 22.2, 13.2. HRMS-ESI (m/z) [M − H]+ calcd for C24H29N2O4 409.2127, found 409.2198.

2-Methoxy-4-((3Z,4E)-3-(2-(pyridin-2-yl)hydrazono)dec-4-en-1-yl)phenol (4c). Orange viscous liquid; yield: 70%; R = 0.65 (100% dichlorometane); IR (neat) υmax 3332 (OH), 1589 (C=N), 1513 (C=C), 1268 (C-O) cm−1.1H NMR (CDCl3, 400 MHz) δ 8.42 (m, 1H), 7.78 (m, 2H), 7.15 (m, 1H), 6.82 (m, 1H), 6.75 (m, 1H), 6.68 (s, 1H), 6.03 (s, 1H), 5.54 (brs, 1H), 3.85 (s, 3H), 3.37 (t, J = 8.0 Hz, 2H), 2.91 (m, 2H), 2.63 (t, J = 8.0 Hz, 2H), 1.64 (m, 2H), 1.36 (m, 4H), 0.98 (m, 3H). 13C NMR (CDCl3, 100 MHz) δ 154.6, 153.6, 147.4, 146.3, 145.2, 143.8, 138.2, 133.5, 121.0, 120.7, 116.3, 114.2, 111.0, 107.0, 55.8, 35.2, 31.7, 30.1, 29.2, 28.3, 22.5, 14.0. HRMS-ESI (m/z) [M + H]+ calcd for C22H30N3O2 368.2338, found 368.2356.

4-((3Z,4E)-3-(2-(Benzo[d]thiazol-2-yl)hydrazono)dec-4-en-1-yl)-2-methoxyphenol (4d). Yellow viscous liquid; yield: 78%; R = 0.40 (100% dichlorometane); IR (neat) υmax 3336 (OH), 1600 (C=N), 1513 (C=C), 1267 (C-O) cm−1.1H NMR (CDCl3, 400 MHz) δ 7.56 (d, J = 8.0 Hz, 2H), 7.40 (d, J = 8.0 Hz, 2H), 7.24 (t, J = 8.0 Hz, 1H), 7.06 (t, J = 8.0 Hz, 1H), 6.75 (d, J = 8.0 Hz, 1H), 6.73 (s, 1H), 6.67 (dd, J = 4.0, 8.0 Hz, 1H), 6.14 (d, J = 16.0 Hz, 1H), 6.02 (m, 1H), 3.80 (s, 3H), 2.71 (brs, 4H), 2.14 (q, J = 8.0 Hz, 2H), 1.39 (m, 2H), 1.25 (m, 4H), 0.84 (m, 3H). 13C NMR (CDCl3, 100 MHz) δ 168.5, 153.6, 149.4, 147.0, 144.3, 136.4, 132.4, 129.7, 129.3, 125.9, 121.9, 121.2, 121.0, 117.8, 114.9, 111.6, 55.8, 32.9, 31.8, 31.3, 29.0, 28.1, 22.4, 13.9. HRMS-ESI (m/z) [M + H]+ calcd for C24H30N3O2S 424.2059, found 424.2212.

4-((3Z,4E)-3-(2-(4H-Imidazol-2-yl)hydrazono)dec-4-en-1-yl)-2-methoxyphenol (4e). Yellow viscous liquid; yield: 72%; R = 0.45 (100% dichlorometane); IR (neat) υmax 3158 (OH), 1657 (C=N), 1611 (C=N), 1513 (C=C), 1274 (C-O) cm−1.1H NMR (CDCl3, 400 MHz) δ 6.81 (s, 1H), 6.71 (m, 1H), 6.64 (m, 1H), 6.01 (m, 2H), 3.82 (s, 3H), 3.59 (brs, 4H), 2.77 (m, 2H), 2.64 (m, 2H), 2.11 (q, J = 8.0 Hz, 2H), 1.36 (m, 2H), 1.24 (m, 4H), 0.83 (m, 3H). 13C NMR (CDCl3, 100 MHz) δ 161.2, 158.2, 147.0, 144.1, 137.1, 132.8, 129.4, 120.8, 114.5, 111.8, 55.8, 42.6, 32.9, 32.3, 31.8, 29.0, 28.4, 22.4, 13.9. HRMS-ESI (m/z) [M + H]+ calcd for C20H31N4O2 359.2447, found 359.2443.

4.3.2. Synthesis of the Pyrazole Derivatives (5a–5k)

To a solution of [6]-shogaol (4) (207 mg, 0.75 mmol) in ethanol (5 mL), phenylhydrazine hydrochloride (108 mg, 0.75 mmol) was added. The solution was refluxed at 80 °C until completion based on TLC. The mixture was filtered, and the residue was washed with ethanol (10 mL) and dried with anhydrous sodium sulfate. Evaporation of the combined solvents gave a crude product. Purification of the crude product by column chromatography (10% ethyl acetate/hexane) gave a dark green viscous liquid of compound 5a (212 mg, 0.58 mmol, 77%).

2-methoxy-4-(2-(5-pentyl-1-phenyl-4,5-dihydro-1H-pyrazol-3-yl)ethyl)phenol (5a). Dark green viscous liquid; yield: 77%; R = 0.65 (70% dichlorometane: hexane); IR (neat) υmax 3336 (OH), 1597 (C=N), 1514 (C=C), 1269 (C-O), 1119 (C-N), 1033 (C-O) cm−1.1H NMR (CDCl3, 400 MHz) δ 7.40 (t, J = 8.0 Hz, 2H), 7.18 (d, J = 8.0 Hz, 2H), 6.97 (d, J = 8.0 Hz, 1H), 6.90 (m, 3H), 5.75 (s, 1H), 4.23 (m, 1H), 3.98 (s, 3H), 3.12 (dd, J = 4.0, 8.0 Hz, 1H), 3.02 (t, J = 8.0 Hz, 2H), 2.82 (t, J = 8.0 Hz, 2H), 2.65 (dd, J = 4.0, 8.0 Hz, 1H), 1.90 (m, 1H), 1.55 (m, 1H), 1.45 (m, 6H), 1.02 (t, J = 8.0 Hz, 3H). 13C NMR (CDCl3, 100 MHz) δ 152.0, 146.3, 145.6, 143.8, 133.1, 128.9, 120.8, 118.2, 114.2, 113.1, 111.0, 59.8, 55.7, 41.1, 32.6, 32.5, 32.4, 31.6, 24.7, 22.6, 13.9. HRMS-ESI (m/z) [M − H]+ calcd for C23H29N2O2 365.2229, found 365.2222.

The same procedure was carried out with o-tolylphenylhydrazine hydrochloride, 2-methoxyphenylhydrazine hydrochloride, 4-methoxyphenylhydrazine hydrochloride, 2-fluorophenylhydrazine hydrochloride, 4-fluorophenylhydrazine hydrochloride, 3-chlorophenylhydrazine hydrochloride, 4-chlorophenylhydrazine hydrochloride, 3-nitrophenylhydrazine hydrochloride, 4-nitrophenylhydrazine hydrochloride, and 4-hydrazinobenzoic acid to convert [6]-shogaol (4) into 5b–5k.

2-methoxy-4-(2-(5-pentyl-1-(o-tolyl)-4,5-dihydro-1H-pyrazol-3-yl)ethyl)phenol (5b). Brown viscous liquid; yield: 80%; R = 0.48 (70% dichlorometane: hexane); IR (neat) υmax 3336 (OH), 1598 (C=N), 1514 (C=C), 1269 (C-O), 1119 (C-N), 1033 (C-O) cm−1.1H NMR (CDCl3, 400 MHz) δ 7.25 (m, 3H), 7.10 (t, J = 8.0 Hz, 1H), 6.92 (d, J = 8.0 Hz, 1H), 6.82 (m, 2H), 5.80 (s, 1H), 4.00 (qd, J = 4.0, 8.0 Hz, 1H), 3.92 (s, 3H), 2.95 (m, 3H), 2.75 (t, J = 8.0 Hz, 2H), 2.58 (dd, J = 4.0, 8.0 Hz, 1H), 2.45 (s, 3H), 1.30 (m, 8H), 0.95 (t, J = 8.0 Hz, 3H). 13C NMR (CDCl3, 100 MHz) δ 153.2, 146.3, 145.5, 143.8, 133.1, 131.9, 130.7, 126.1, 123.7, 121.2, 120.7, 114.2, 111.0, 64.4, 55.7, 40.7, 32.6, 32.5, 31.6, 31.4, 25.7, 22.5, 19.1, 13.8. HRMS-ESI (m/z) [M − H]+ calcd for C24H31N2O2 379.2386, found 379.2353.

2-methoxy-4-(2-(1-(2-methoxyphenyl)-5-pentyl-4,5-dihydro-1H-pyrazol-3-yl)ethyl) phenol (5c). Brown viscous liquid; yield: 82%; R = 0.40 (70% dichlorometane: hexane); IR (neat) υmax 3330 (OH), 1594 (C=N), 1513 (C=C), 1232 (C-O), 1121 (C-N), 1036 (C-O) cm−1.1H NMR (CDCl3, 400 MHz) δ 7.20 (d, J = 8.0 Hz, 1H), 6.85 (m, 1H), 6.75 (m, 3H), 6.60 (m, 2H), 5.45 (s, 1H), 4.25 (m, 1H), 3.75 (s, 3H), 2.75 (m, 3H), 2.55 (t, J = 8.0 Hz, 2H), 2.35 (dd, J = 4.0, 8.0 Hz, 1H), 1.10 (m, 8H), 0.70 (t, J = 8.0 Hz, 3H). 13C NMR (CDCl3, 100 MHz) δ 153.0, 150.2, 146.3, 143.8, 135.7, 133.2, 122.8, 121.1, 121.0, 120.8, 114.2, 111.0, 110.9, 62.8, 55.8, 55.4, 40.4, 32.8, 32.5, 31.5, 30.8, 25.0, 22.4, 13.9. HRMS-ESI (m/z) [M − H]+ calcd for C24H31N2O3 395.2335, found 395.2306.

2-methoxy-4-(2-(1-(4-methoxyphenyl)-5-pentyl-4,5-dihydro-1H-pyrazol-3-yl)ethyl) phenol (5d). Brown viscous liquid; yield: 79%; R = 0.40 (70% dichlorometane: hexane); IR (neat) υmax 3336 (OH), 1607 (C=N), 1512 (C=C), 1247 (C-O), 1120 (C-N), 1031 (C-O) cm−1.1H NMR (CDCl3, 400 MHz) δ 7.20 (d, J = 8.0 Hz, 2H), 7.05 (m, 3H), 6.95 (m, 2H), 5.78 (s, 1H), 4.10 (s, 1H), 4.08 (s, 3H), 4.05 (s, 3H), 3.98 (s, 3H), 3.15 (dd, J = 4.0, 8.0 Hz, 1H), 3.08 (t, J = 8.0 Hz, 2H), 2.85 (t, J = 8.0 Hz, 2H), 2.70 (dd, J = 4.0, 8.0 Hz, 1H), 1.95 (m, 2H), 1.50 (m, 6H), 1.10 (t, J = 8.0 Hz, 3H). 13C NMR (CDCl3, 100 MHz) δ 153.4, 152.2, 146.4, 143.8, 140.7, 133.2, 120.8, 116.1, 114.4, 114.2, 111.0, 62.2, 55.8, 55.6, 41.3, 32.7, 32.7, 32.5, 31.7, 25.2, 22.6, 14.0. HRMS-ESI (m/z) [M – H]+ calcd for C24H31N2O3 395.2335, found 395.2307.

4-(2-(1-(2-fluorophenyl)-5-pentyl-4,5-dihydro-1H-pyrazol-3-yl)ethyl)-2-methoxyphenol (5e). Brown viscous liquid; yield: 82%; R = 0.55 (70% dichlorometane: hexane); IR (neat) υmax 3336 (OH), 1610 (C=N), 1514 (C=C), 1269 (C-O), 1233 (C-F), 1118 (C-N), 1033 (C-O) cm−1. 1H NMR (CDCl3, 400 MHz) δ 7.60 (t, J = 8.0 Hz, 1H), 7.18 (m, 2H), 7.00 (m, 2H), 6.90 (m, 2H), 5.90 (s, 1H), 4.23 (m, 1H), 3.98 (s, 3H), 3.08 (m, 1H), 3.05 (t, J = 8.0 Hz, 2H), 2.85 (t, J = 8.0 Hz, 2H), 2.68 (dd, J = 4.0, 8.0 Hz, 1H), 1.55 (m, 1H), 1.40 (m, 7H), 1.00 (t, J = 8.0 Hz, 3H). 13C NMR (CDCl3, 100 MHz) δ 153.4, 150.7, 146.4, 143.8, 134.0, 132.9, 124.3, 121.3, 120.7, 120.1, 115.7, 114.2, 110.9, 62.3, 55.7, 40.5, 32.6, 32.2, 31.4, 31.2, 24.6, 22.4, 13.8. HRMS-ESI (m/z) [M − H]+ calcd for C23H28FN2O2 383.2135, found 383.2121.

4-(2-(1-(4-fluorophenyl)-5-pentyl-4,5-dihydro-1H-pyrazol-3-yl)ethyl)-2-methoxyphenol (5f). Brown viscous liquid; yield: 80%; R = 0.55 (70% dichlorometane: hexane); IR (neat) υmax 3336 (OH), 1605 (C=N), 1506 (C=C), 1269 (C-O), 1219 (C-F), 1120 (C-N), 1033 (C-O) cm−1. 1H NMR (CDCl3, 400 MHz) δ 7.18 (m, 4H), 7.05 (d, J = 8.0 Hz, 1H), 6.95 (m, 2H), 5.90 (s, 1H), 4.20 (m, 1H), 4.05 (s, 3H), 3.18 (m, 1H), 3.10 (t, J = 8.0 Hz, 2H), 2.88 (t, J = 8.0 Hz, 2H), 2.72 (dd, J = 4.0, 8.0 Hz, 1H), 1.95 (m, 1H), 1.65 (m, 1H), 1.50 (m, 6H), 1.10 (t, J = 8.0 Hz, 3H). 13C NMR (CDCl3, 100 MHz) δ 152.4, 146.4, 143.8, 133.0, 120.8, 115.4, 115.2, 114.8, 114.7, 114.2, 111.0, 60.9, 55.7, 41.2, 32.6, 32.4, 32.3, 31.6, 24.9, 22.5, 13.9. HRMS-ESI (m/z) [M − H]+ calcd for C23H28 FN2O2 383.2135, found 383.2122.

4-(2-(1-(3-chlorophenyl)-5-pentyl-4,5-dihydro-1H-pyrazol-3-yl)ethyl)-2-methoxyphenol (5g). Dark viscous liquid; yield: 75%; R = 0.60 (70% dichlorometane: hexane); IR (neat) υmax 3336 (OH), 1592 (C=N), 1513 (C=C), 1269 (C-O), 1233 (C-N), 1033 (C-O) cm−1. 1H NMR (CDCl3, 400 MHz) δ 7.25 (t, J = 8.0 Hz, 1H), 7.20 (d, J = 2.0 Hz, 1H), 6.98 (d, J = 8.0 Hz, 1H), 6.95 (dd, J = 2.0, 8.0 Hz, 1H), 6.90 (d, J = 2.0 Hz, 1H), 6.85 (dd, J = 2.0, 8.0 Hz, 2H), 5.70 (s, 1H), 4.20 (m, 1H), 3.98 (s, 3H), 3.10 (dd, J = 4.0, 8.0 Hz, 1H), 3.00 (t, J = 8.0 Hz, 2H), 2.80 (t, J = 8.0 Hz, 2H), 2.62 (dd, J = 4.0, 8.0 Hz, 1H), 1.85 (m, 1H), 1.55 (m, 1H), 1.40 (m, 6H), 1.00 (t, J = 8.0 Hz, 3H). 13C NMR (CDCl3, 100 MHz) δ 152.8, 146.3, 146.3, 143.8, 134.7, 132.9, 129.9, 120.7, 117.6, 114.2, 112.7, 111.0, 110.5, 59.3, 55.7, 41.1, 32.4, 32.2, 32.1, 31.5, 24.4, 22.5, 13.9. HRMS-ESI (m/z) [M − H]+ calcd for C23H28ClN2O2 399.1839, found 399.1802.

4-(2-(1-(4-chlorophenyl)-5-pentyl-4,5-dihydro-1H-pyrazol-3-yl)ethyl)-2-methoxyphenol (5h). Dark viscous liquid; yield: 83%; R = 0.62 (70% dichlorometane: hexane); IR (neat) υmax 3336 (OH), 1596 (C=N), 1514 (C=C), 1269 (C-O), 1233 (C-N), 1033 (C-O), 816 (C-Cl) cm−1. 1H NMR (CDCl3, 400 MHz) δ 7.20 (d, J = 8.0 Hz, 2H), 6.95 (d, J = 8.0 Hz, 2H), 6.88 (d, J = 8.0 Hz, 1H), 6.78 (d, J = 2.0 Hz, 1H), 6.75 (d, J = 2.0, 8.0 Hz, 1H), 5.65 (s, 1H), 4.05 (m, 1H), 3.85 (s, 3H), 3.00 (dd, J = 4.0, 8.0 Hz, 1H), 2.90 (t, J = 8.0 Hz, 2H), 2.68 (t, J = 8.0 Hz, 2H), 2.52 (dd, J = 2.0, 8.0 Hz, 1H), 1.75 (m, 1H), 1.40 (m, 1H), 1.30 (m, 6H), 0.90 (t, J = 8.0 Hz, 3H). 13C NMR (CDCl3, 100 MHz) δ 152.5, 146.3, 144.0, 143.8, 133.0, 128.7, 122.7, 120.8, 114.2, 114.0, 110.9, 59.6, 55.7, 41.2, 32.5, 32.2, 32.2, 31.5, 24.6, 22.5, 13.9. HRMS-ESI (m/z) [M − H]+ calcd for C23H28ClN2O2 399.1839, found 399.1809.

2-methoxy-4-(2-(1-(3-nitrophenyl)-5-pentyl-4,5-dihydro-1H-pyrazol-3-yl)ethyl)phe nol (5i). Brown viscous liquid; yield: 70%; R = 0.35 (70% dichlorometane: hexane); IR (neat) υmax 3508 (OH), 1614 (C=N), 1514 (C=C), 1491 (N-O), 1463 (Ar), 1269 (C-O), 1233 (C-N), 1032 (C-O) cm−1. 1H NMR (CDCl3, 400 MHz) δ 7.70 (s, 1H), 7.50 (dd, J = 4.0, 8.0 Hz, 1H), 7.25 (t, J = 8.0 Hz, 1H) 7.18 (d, J = 8.0 Hz, 1H), 6.78 (d, J = 8.0 Hz, 1H), 6.70 (s, 1H), 6.65 (d, J = 8.0 Hz, 1H), 5.48 (s, 1H), 4.10 (m, 1H), 3.80 (s, 3H), 3.00 (dd, J = 4.0, 8.0 Hz, 1H), 2.85 (t, J = 8.0 Hz, 2H), 2.60 (t, J = 8.0 Hz, 2H), 2.50 (dd, J = 2.0, 8.0 Hz, 1H), 1.65 (m, 1H), 1.35 (m, 1H), 1.20 (m, 6H), 0.85 (t, J = 8.0 Hz, 3H). 13C NMR (CDCl3, 100 MHz) δ 153.8, 149.2, 146.4, 145.7, 143.9, 132.8, 129.5, 120.8, 118.1, 114.2, 112.1, 110.9, 106.7, 59.1, 55.8, 41.3, 32.5, 32.2, 31.9, 31.5, 24.3, 22.5, 13.9. HRMS-ESI (m/z) [M + H]+ calcd for C23H30N3O4 412.2236, found 412.2203.

2-methoxy-4-(2-(1-(4-nitrophenyl)-5-pentyl-4,5-dihydro-1H-pyrazol-3-yl)ethyl)phe nol (5j). Brown viscous liquid; yield: 78%; R = 0.35 (70% dichlorometane: hexane); IR (neat) υmax 3433 (OH), 1593 (C=N), 1512 (C=C), 1488 (N-O), 1273 (C-O), 1179 (C-N), 1108 (C-O) cm−1. 1H NMR (CDCl3, 400 MHz) δ 8.38 (d, J = 8.0 Hz, 2H), 7.18 (d, J = 8.0 Hz, 2H), 7.10 (d, J = 8.0 Hz, 1H) 7.00 (m, 2H), 5.80 (s, 1H), 4.50 (m, 1H), 4.10 (s, 3H), 3.30 (dd, J = 4.0, 8.0 Hz, 1H), 3.18 (t, J = 8.0 Hz, 2H), 2.95 (dt, J = 2.0, 8.0 Hz, 2H), 2.82 (dd, J = 2.0, 8.0 Hz, 1H), 1.95 (m, 1H), 1.65 (m, 1H), 1.50 (m, 6H), 1.15 (t, J = 8.0 Hz, 3H). 13C NMR (CDCl3, 100 MHz) δ 156.7, 148.6, 146.4, 144.0, 137.9, 132.5, 126.2, 120.8, 114.3, 110.9, 110.7, 58.4, 55.8, 41.3, 32.4, 32.2, 31.9, 31.4, 24.2, 22.5, 13.9. HRMS-ESI (m/z) [M − H]+ calcd for C23H38N3O4 410.2080, found 410.2039.

4-(3-(4-hydroxy-3-methoxyphenethyl)-5-pentyl-4,5-dihydro-1H-pyrazol-1-yl)benzo ic acid (5k). Brown viscous liquid; yield: 71%; R = 0.40 (2% methanol: dichlorometane); IR (neat) υmax 3400 (OH), 1669 (C = O), 1595 (C=N), 1514 (C=C), 1267 (C-O), 1169 (C-N), 1120 (C-O) cm−1.1H NMR (CDCl3, 400 MHz) δ 8.20 (d, J = 8.0 Hz, 2H), 7.18 (d, J = 8.0 Hz, 2H), 7.05 (d, J = 8.0 Hz, 1H), 6.95 (m, 2H), 4.40 (m, 1H), 4.05 (s, 3H), 3.20 (dd, J = 4.0, 8.0 Hz, 1H), 3.10 (t, J = 8.0 Hz, 2H), 2.88 (t, J = 8.0 Hz, 2H), 2.72 (dd, J = 4.0, 8.0 Hz, 1H), 1.95 (m, 1H), 1.65 (m, 1H), 1.50 (m, 6H), 1.10 (t, J = 8.0 Hz, 3H). 13C NMR (CDCl3, 100 MHz) δ 172.3, 154.6, 148.4, 146.4, 143.9, 132.8, 132.0, 120.8, 117.3, 114.3, 111.1, 111.0, 58.4, 55.8, 41.1, 32.5, 32.3, 32.0, 31.5, 24.3, 22.5, 13.9. HRMS-ESI (m/z) [M + H]+ calcd for C24H31N2O4 411.2284, found 411.2244.

4.3.3. Synthesis of the Amino Derivatives (6a–6c, 7)

To a solution of [6]-shogaol (4) (90 mg, 0.32 mmol) in ethanol (5 mL), 2-aminothiophenol (60 mg, 0.48 mmol) was added at room temperature. The mixture was refluxed at 80 °C. After completion based on TLC, the mixture was filtered, and the residue was washed with ethanol (10 mL) and dried with anhydrous sodium sulfate. Evaporation of the combined solvents gave crude product. Purification of the crude product by column chromatography (5% MeOH in CH2Cl2) gave a yellow viscous liquid of compound 6a (92 mg, 0.25 mmol, 78%).

10-(4-Hydroxy-3-methoxyphenyl)-6-((2-mercaptophenyl)amino)decan-8-one (6a). Yellow viscous liquid; yield: 78%; R = 0.55 (100% dichlorometane); IR (neat) υmax 3359 (OH), 1710 (C = O), 1512 (C=C), 1267 (C-O) cm−1.1H NMR (CDCl3, 400 MHz) δ 7.52 (d, J = 8.0 Hz, 1H), 7.36 (t, J = 8.0 Hz, 1H), 7.12 (d, J = 8.0 Hz, 1H), 7.03 (t, J = 8.0 Hz, 1H), 6.85 (d, J = 8.0 Hz, 1H), 6.81 (d, J = 4.0 Hz, 1H), 6.76 (dd, J = 4.0, 8.0 Hz, 1H), 3.84 (s, 3H), 3.71 (m, 1H), 3.04 (m, 2H), 2.87 (m, 2H), 2.39 (dd, J = 4.0, 12.0 Hz, 1H), 2.16 (dd, J = 4.0, 12.0 Hz, 1H), 1.57 (m, 2H), 1.26 (m, 6H), 0.89 (m, 3H). 13C NMR (CDCl3, 100 MHz) δ 208.3, 146.4, 143.9, 137.5, 135.3, 133.4, 129.4, 124.6, 124.2, 122.7, 120.9, 114.3, 111.2, 57.0, 55.9, 43.0, 39.2, 38.0, 32.1, 29.3, 26.6, 22.5, 14.0. HRMS-ESI (m/z) [M − H2O + H]+ calcd for C23H30NO2S 384.1997, found 384.1917.

The same procedure was conducted with 2-aminophenol, aniline, and o-phenylenediamine to convert 6-shogaol (4) into 6b, 6c and 7.

10-(4-Hydroxy-3-methoxyphenyl)-6-((2-hydroxyphenyl)amino)decan-8-one (6b). Orange viscous liquid; yield: 75%; R = 0.40 (100% dichlorometane); IR (neat) υmax 3312 (OH), 1709 (C = O), 1508 (C=C), 1269 (C-O) cm−1.1H NMR (CDCl3, 400 MHz) δ 7.74 (d, J = 8.0 Hz, 1H), 7.39 (m, 3H), 6.78 (d, J = 8.0 Hz, 1H), 6.64 (m, 2H), 6.40 (s, 1H), 6.22 (s, 1H), 5.82 (d, J = 8.0 Hz, 1H), 5.54 (brs, 1H), 3.94 (m, 1H), 3.83 (s, 3H), 2.81 (t, J = 8.0 Hz, 2H), 2.71 (m, 3H), 2.59 (dd, J = 8.0, 16.0 Hz, 1H), 1.58 (m, 2H), 1.25 (m, 6H), 0.85 (t, J = 8.0 Hz, 3H). 13C NMR (CDCl3, 100 MHz) δ 207.7, 146.4, 144.7, 143.9, 142.6, 132.6, 129.0, 128.4, 125.3, 120.8, 116.0, 114.4, 111.0, 55.8, 48.7, 48.9, 45.5, 34.6, 31.5, 29.3, 25.8, 22.5, 14.0. HRMS-ESI (m/z) [M + 2H]+ calcd for C23H33NO4 387.2410, found 387.2668.

10-(4-Hydroxy-3-methoxyphenyl)-6-(phenylamino)decan-8-one (6c). Yellow viscous liquid; yield: 74%; R = 0.65 (100% dichloro-metane); IR (neat) υmax 3390 (OH), 1705 (C = O), 1512 (C=C), 1266 (C-O) cm−1.1H NMR (CDCl3, 400 MHz) δ 7.17 (d, J = 8.0 Hz, 1H), 7.16 (d, J = 8.0 Hz, 1H), 6.81 (d, J = 8.0 Hz, 1H), 6.69 (m, 1H), 6.64 (s, 1H), 6.63 (m, 1H),6.58 (m, 1H), 3.85 (s, 3H), 3.84 (m, 1H), 2.79 (t, J = 8.0 Hz, 2H), 2.68 (t, J = 8.0 Hz, 2H), 2.63 (m, 1H), 2.55 (dd, J = 8.0, 16.0 Hz, 1H), 1.51 (m, 2H), 1.26 (m, 6H), 0.88 (m, 3H). 13C NMR (CDCl3, 100 MHz) δ 209.6, 147.2, 146.4, 143.9, 132.9, 129.4, 120.7, 117.4, 114.3, 113.4, 111.0, 55.8, 49.9, 47.5, 45.6, 35.3, 31.9, 29.3, 26.3, 22.7, 14.1. HRMS-ESI (m/z) [M + H]+ calcd for C23H32NO3 370.2382, found 370.2385.

2-Methoxy-4-(2-(2-pentyl-2,3-dihydro-1H-benzo[b][1,4]diazepin-4-yl)ethyl)phenol (7). Yellow viscous liquid; yield: 70%; R = 0.45 (100% dichlorometane); IR (neat) υmax 3727 (NH), 3292 (OH), 1704 (C = O), 1512 (C=C), 1230 (C-O) cm−1.1H NMR (CDCl3, 400 MHz) δ 7.17 (dd, J = 4.0, 8.0 Hz, 1H), 6.97 (m, 2H), 6.84 (d, J = 8.0 Hz, 1H), 6.80 (d, J = 4.0 Hz, 1H), 6.73 (m, 2H), 3.85 (s, 3H), 3.84 (m, 1H), 2.98 (t, J = 8.0 Hz, 2H), 2.82 (t, J = 8.0 Hz, 2H), 2.41 (dd, J = 4.0, 12.0 Hz, 1H), 2.22 (dd, J = 8.0, 16.0 Hz, 1H), 1.52 (m, 2H), 1.31 (m, 6H), 0.89 (t, J = 8.0 Hz, 3H). 13C NMR (CDCl3, 100 MHz) δ 174.1, 146.4, 143.9, 139.3, 138.1, 133.5, 127.6, 125.7, 121.3, 121.0, 114.3, 114.3, 111.2, 65.7, 55.9, 44.0, 38.3, 37.7, 32.2, 31.7, 25.6, 22.6, 14.0. HRMS-ESI (m/z) [M + H]+ calcd for C23H31N2O2 367.2386, found 367.2385.

4.4. HDAC Activity Assay

The semi-synthetic derivatives were evaluated for their ability to inhibit HDAC enzymes. Inhibition of HDAC activity in vitro was assessed using the Fluor-de-Lys HDAC activity assay kit (Biomol, Enzo Life Sciences International, Inc., Farmingdale, NY, USA). The HeLa nuclear extract provided with the kit was used as a source of HDAC enzymes for the in vitro study. TSA was used as the positive control. The HeLa nuclear extract, substrate, buffer and inhibitors were incubated. Deacetylation of the substrate was performed next by adding a developer to generate a fluorophore. The spectra Max Gemini XPS microplate spectrofluorometer (Molecular Devices, San Jose, CA, USA) was used to measure fluorescence signal with excitation at 360 nm and emission at 460 nm. A decrease in fluorescence signal indicated an inhibition of HDAC activity. Trichostatin A (TSA) was used as a positive control. All experiments were carried out in triplicate.

4.5. MTT Assay

The MTT reduction assay was performed with non-cancer (Vero), human cervical cancer (HeLa), human colon cancer (HCT116), human breast adenocarcinoma cancer (MCF-7), and cholangiocarcinoma (H-69 (Vero), KKU-100 and KKU-M213B) cell lines according to the previously described method [34,35]. Briefly, cells were seeded in 96-well plates. The next day, cells were exposed to the selected compounds at various concentrations and incubated for 24, 48 and 72 h. After incubation, the culture medium was exchanged with 110 µL of MTT (0.5 mg/mL in PBS medium) and further incubated for 2 h. The amount of MTT formazan product was determined after dissolving in DMSO by measuring its absorbance with a microplate reader (Bio-Rad Laboratories, Hercules, CA, USA) at a test wavelength of 550 nm and a reference wavelength of 655 nm. The cell viability was expressed as a percentage of the viable cells of control culture conditions, and the IC50 values of each group were calculated.

4.6. Molecular Docking Studies

The crystal structures of HDAC1, HDAC2, HDAC3 and HDAC8 (PDB entry code: 4BKX, 3MAX, 4A69 and 1T64, respectively) were obtained from the Protein Data Bank (http://www.rcsb.org/pdb (accessed on 1 March 2022). All water and non-interacting ions as well as ligands were removed. Then, all missing hydrogen and side-chain atoms were added using the ADT program. Gasteiger charges were calculated for the system. For ligand setup, the molecular modeling program Gaussview was used to build the ligands. These ligands were optimized with the AM1 level by using Gaussian03W. Molecular docking studies were performed for 50 runs using AutoDockTools 1.5.4 (ADT) (La Jolla, CA, USA) and AutoDock 4.2 programs and Lamarckian genetic algorithm search. A grid box size of 60 × 60 × 60 points with a spacing of 0.375 Å between the grid points was implemented and covered almost the entire HDAC protein surface. For TSA and other inhibitors, the single bonds were treated as active torsional bonds.